Plant Tissue Culture.pdf

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Plant Tissue

Culture:

An Introductory

Text

Sant Saran Bhojwani

Prem Kumar Dantu

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Plant Tissue Culture:

An Introductory Text

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Sant Saran Bhojwani

Prem Kumar Dantu

Plant Tissue Culture:

An Introductory Text

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Sant Saran Bhojwani Prem Kumar Dantu Department of Botany

Dayalbagh Educational Institute Agra, Uttar Pradesh

India

ISBN 978-81-322-1025-2 ISBN 978-81-322-1026-9 (eBook) DOI 10.1007/978-81-322-1026-9

Springer New Delhi Heidelberg New York Dordrecht London Library of Congress Control Number: 2012954643

Ó Springer India 2013

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law.

The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein.

Printed on acid-free paper

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Dedicated to the most Revered

Dr. M. B. Lal Sahab (1907–2002)

D.Sc. (Lucknow), D.Sc. (Edinburgh),

the visionary Founder Director of the

Dayalbagh Educational Institute, for

the inspiration and strength to

undertake and complete the task of

writing this book

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Preface

Plant tissue culture (PTC) broadly refers to cultivation of plant cells, tissues, organs, and plantlets on artiﬁcial medium under aseptic and controlled environmental conditions. PTC is as much an art as a science. It is the art of growing experimental plants, selecting a suitable plant organ or tissue to initiate cultures, cleaning, sterilization and trimming it to a suitable size, and planting it on a culture medium in right orien-tation while maintaining complete asepsis. It also requires an experi-enced and vigilant eye to select healthy and normal tissues for subculture. PTC involves a scientiﬁc approach to systematically opti-mize physical (nature of the substrate, pH, light, temperature and humidity), chemical (composition of the culture medium, particularly nutrients and growth regulators), biological (source, physiological status and size of the explant), and environmental (gaseous environment inside the culture vial) parameters to achieve the desired growth rate, cellular metabolism, and differentiation.

The most important contribution made through PTC is the demon-stration of the unique capacity of plant cells to regenerate full plants, via organogenesis or embryogenesis, irrespective of their source (root, leaf, stem, ﬂoral parts, pollen, endosperm) and ploidy level (haploid, diploid, triploid). PTC is also the best technique to exploit the cellular totipo-tency of plant cells for numerous practical applications, and offers technologies for crop improvement (haploid and triploid production, in vitro fertilization, hybrid embryo rescue, variant selection), clonal propagation (Micropropagation), virus elimination (shoot tip culture), germplasm conservation, production of industrial phytochemicals, and regeneration of plants from genetically manipulated cells by recombi-nant DNA technology (genetic engineering) or cell fusion (somatic hybridization). PTC has been extensively employed for basic studies related to plant physiology (photosynthesis, nutrition of plant cells, and embryos), biochemistry, cellular metabolism, morphogenesis (organo-genesis, embryogenesis), phytopathology (plant microbe interaction), histology (cytodifferentiation), cytology (cell cycle), etc. Indeed the discovery of ﬁrst cytokinin is based on PTC studies.

Thus, PTC is an exciting area of basic and applied sciences with considerable scope for further research. Considerable work is being done to understand the physiology and genetics of embryogenesis and

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organogenesis using PTC systems, especially Arabidopsis and carrot, which are likely to enhance the efﬁciency of in vitro regeneration pro-tocols. Therefore, PTC forms a part of most of the courses on plant sciences (Developmental Botany, Embryology, Physiology, Genetics, Plant Breeding, Horticulture, Sylviculture, Phytopathology, etc.) and is an essential component of Plant Biotechnology.

After the ﬁrst book on ‘‘Plant Tissue Culture’’ by Prof. P. R. White in 1943, several volumes describing different aspects of PTC have been published. Most of these are compilations of invited articles by different experts or proceedings of conferences. More recently, a number of books describing the methods and protocols for one or more techniques of PTC have been published which should serve as useful laboratory manuals. The impetus for writing this book was to make available an up-to-date text covering all theoretical and practical aspects of PTC for the students and early career researchers of plant sciences and agricul-tural biotechnology. The book includes 19 chapters profusely illustrated with half-tone pictures and self-explanatory diagrams. Most of the chapters include relevant media compositions and protocols that should be helpful in conducting laboratory exercises. For those who are inter-ested in further details, Sugginter-ested Further Reading are given at the end of each chapter. We hope that the readers will ﬁnd it useful. Suggestions for further improvement of the book are most welcome.

During the past two decades or so research in the area of plant biotechnology has become a closed door activity because many renowned scientists have moved from public research laboratories in universities and institutions to the private industry. Consequently, detailed information on many recent developments is not readily available.

We would like to thank many scientists who provided illustrations from their works and those who have helped us in completing this mammoth task. The help of Mr. Jai Bhargava and Mr. Atul Haseja in preparing some of the illustrations is gratefully acknowledged.

October 2012 Sant Saran Bhojwani Prem Kumar Dantu

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About the Book

Plant tissue culture (PTC) is basic to all plant biotechnologies and is an exciting area of basic and applied sciences with considerable scope for further research. PTC is also the best approach to demonstrate the totipotency of plant cells, and to exploit it for numerous practical applications. It offers technologies for crop improvement (haploid and triploid production, in vitro fertilization, hybrid embryo rescue, variant selection), clonal propagation (micropropagation), virus elimination (shoot tip culture), germplasm conservation, production of industrial phytochemi-cals, and regeneration of plants from genetically manipulated cells by recombinant DNA technology (genetic engineering) or cell fusion (somatic hybridization and cybridization). Considerable work is being done to understand the physiology and genetics of in vitro embryogenesis and organogenesis using model systems, especially Arabidopsis and carrot, which is likely to enhance the efficiency of in vitro regeneration protocols. All these aspects are covered extensively in this book.

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1 Historical Sketch

Gottlieb Haberlandt, a German botanist, made the first attempts to culture fully differentiated single cells isolated from the leaves of Lamium purpureum, petioles of Eichhornia crassipes, glandular hairs of Pulmonaria mollissima, and stamen hairs of Tradescantia in a simple nutrient solution of Knop. The purpose of this experi-ment was to achieve divisions in these cells and obtain complete plants from them to verify the concept of cellular totipotency inherent in the famous Cell Theory put forward by Schleiden (1838) and Schwann (1839). The cultured cells survived for up to 1 month and also increased in volume but did not divide. Although Haberlandt could not achieve his goals, his genius is apparent in his classic paper presented before the Vienna Academy of Science in Berlin in 1902 wherein he laid down, for the first time, several postulates and principles of plant tissue culture. He had proposed that cells in the plant body stop growing after acquiring the features required by the entire organism without losing their (cell’s) inherent potentiality for further growth and are capable of resuming uninterrupted growth on getting suitable stimulus. He also put forward the view that it should be possible to obtain embryos from vegetative cells. With the passage of time, most of the postulates of Haberlandt have been confirmed experimentally, and there-fore he is justifiably recognized as the father of plant tissue culture.

A new line of investigation was initiated by Hannig (1904) that later emerged as an important applied area of plant tissue culture. He excised nearly mature embryos of some crucifers and successfully cultured them to maturity on min-eral salts and sugar solution. In 1925, Laibach made a very significant contribution when he demonstrated that in the cross Linum perenne x L. austriacum the hybrid embryos, which nor-mally abort prematurely, could be rescued to obtain full hybrid plants by excising them from the immature seeds and culturing on nutrient medium. Embryo culture has since become a useful tool in the hands of plant breeders to obtain rare hybrids which otherwise fail due to post-zygotic sexual incompatibility (Chap. 11). Van Overbeek et al. (1940) demonstrated for the

GOTTLIEB HABERLANDT (1854-1945)

S. S. Bhojwani and P. K. Dantu, Plant Tissue Culture: An Introductory Text, DOI: 10.1007/978-81-322-1026-9_1,Ó Springer India 2013

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first time, the stimulatory effect of coconut milk on development of young embryos of Datura. It was possible only in 1993 that as small as 8-celled embryos of Brassica juncea could be cultured successfully using double-layer culture system and a complex nutrient medium (Liu et al. 1993). Almost the same time, Kranz and Lörz (1993) and Holm et al. (1994) succeeded in in vitro cultivation of excised in vitro and in vivo formed zygotes, respectively. However, this required the use of a nurse tissue.

In 1922, Kotte in Germany and Robbins in the USA suggested that the meristematic cells in shoot buds and root tips could possibly be used to initiate in vitro cultures. Their work on root cul-ture, although not very successful, opened up a new approach to tissue culture studies. In 1932, White started his famous work on isolated root culture, and in 1934 he announced the establish-ment of continuously growing root cultures of tomato. Some of these root cultures were main-tained, by periodic subcultures, until shortly before his death in 1968, in India. The medium initially used by White contained inorganic salts, yeast extract, and sugar. Yeast extract was later replaced with the three B vitamins, namely pyri-doxine, thiamine, and nicotinic acid. This her-alded the first synthetic medium, which was widely used as basal medium for a variety of cell and tissue cultures. During 1939–1950, Street and his students extensively worked on the root culture system to understand the importance of vitamins in plant growth and root-shoot relationship. The other postulate of Kotte and Robbins was realized when Loo (1945) estab-lished excellent cultures of Asparagus and Cuscuta shoot tips. Finally, Ball (1946) suc-ceeded in raising whole plants from shoot tip (apical meristem plus a couple of leaf primordia) cultures of Lupinus and Tropaeolum.

The discovery of auxin (Kogl et al. 1934) and recognition of the importance of B vitamins in plant growth (White 1937) gave the required impetus for further progress in the field of plant tissue culture. Using indoleacetic acid and B vitamins, Gautheret (1939) obtained continu-ously growing cultures from carrot root cambium. In the same year, White (1939) and Nobécourt

(1939) reported the establishment of callus cul-tures from tumor tissue of the hybrid Nicotiana glauca 9 Nicotiana langsdorffii and carrot, respectively. These three scientists are credited for laying the foundation for further work in the field of plant tissue culture. The methods and media now used are, in principle, modifications of those established by these three pioneers in 1939. The first book on plant tissue culture, authored by White, was published in 1943.

PHILIP R. WHITE (1901-1968) ROGER J. GAUTHERET (1910-1997) PIERRE NOBÉCOURT (1895-1961) 2 1 Historical Sketch

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During 1950s Skoog and his co-workers, at the University of Wisconsin, USA made several major contributions toward the progress of plant tissue culture. Jablonski and Skoog (1954) tested several plant extracts to induce divisions in mature pith cells of tobacco and found yeast extract to be most suitable in this respect. Miller et al. (1955) isolated the first cell division factor from degraded sample of herring sperm and named it 6-furfurylamino purine, commonly called kinetin. Following this discovery, several natural and synthetic cytokinins were identified, of which benzylamino purine (BAP) is most widely used in plant tissue cultures. The avail-ability of cytokinins made it possible to induce divisions in cells of highly mature and differ-entiated tissues, such as mesophyll and endo-sperm from dried seeds. With the discovery of auxins and cytokinins the stage was set for rapid developments in the field of plant tissue culture. The classic experiments of Skoog and Miller (1957) demonstrated chemical regulation of organogenesis in tobacco tissue cultures by manipulating auxin and kinetin ratio in the medium (Chap. 6). Relatively high concentra-tion of auxin promoted rooting whereas higher levels of cytokinin favored shoot bud differen-tiation. In 1962, Murashige and Skoog formu-lated the now most extensively used plant tissue culture medium, popularly called MS medium. It contains 25 times higher salt concentration than the Knop’s medium, particularly in NO3

-and NH4+ions (Thorpe2007).

The dream of Haberlandt of cultivating isolated single cells began to be realized with the work of Muir. In 1953, Muir demonstrated that by trans-ferring callus tissues to liquid medium and agitat-ing the cultures on a shakagitat-ing machine, it was possible to break the tissues into small cell aggre-gates and single cells. Muir et al. (1954) succeeded in inducing the single cells to divide by placing them individually on separate filter papers, resting on the top of well-established callus cultures that acted as a nurse tissue, and supplied the necessary factors for cell division. Jones et al. (1960) designed a microchamber method for growing single cells in hanging drops of a conditioned medium (medium in which tissue has been grown for some time). This technique allowed continuous observation of the cultured cells. Using this tech-nique, Vasil and Hildebrandt (1965) were able to raise complete plants starting from single cells of tobacco. An important biological technique of cloning large number of single cells was, however, developed in 1960 by Bergmann. It involved mixing single cell suspension with warm, molten agar medium, and plating the cells in a Petri dish where the medium solidified. This cell plating technique is now widely used for cloning cells (Chap. 4) and protoplast culture experiments (Chap. 14). The work of Kohlenbach (1966) came closest to the experiment of Haberlandt. He successfully cultured mature mesophyll cells of Macleaya cordata and obtained germinable somatic embryos (Lang and Kohlenbach 1975). Kohlenbach is also credited for providing con-vincing evidence that an isolated fully differenti-ated mesophyll cell of Zinnia elegans can directly differentiate(transdifferentiation) into a tracheary

FOLKE SKOOG (1908-2001)

TOSHIO MURASHIGE (Born 1930)

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element without cell division (Kohlenbach and Schmidt 1975). This provided a model system for detailed cytological, molecular, and genetic stud-ies on the differentiation of tracheary elements by Komamine and his students (Chap. 5).

White (1934) during the course of his work with virus-infected roots observed that some of the subcultures were free of viruses. Limasset and Cornuet (1949) verified that lack of viruses in the meristematic cells is true not only for root tips but also for shoot tips. Taking a cue from this, Morel and Martin (1952) raised virus-free plants of Dahlia by meristem culture of infected plants. Shoot tip culture, alone or in combination with chemotherapy or/and thermotherapy, has since become the most popular technique to obtain virus-free plants from infected stocks (Chap. 16).

While applying the technique of shoot tip culture for raising virus-free individuals of an orchid, Morel (1960) realized the potential of this method for rapid clonal propagation. The tech-nique allowed the production of almost 4 million genetically identical plants from a single bud in 1 year. This revolutionized the orchid industry, which was dependent on seeds for multiplication. This method of in vitro clonal propagation, pop-ularly called micropropagation, was soon exten-ded, with modifications, to other angiosperms. Toshio Murashige (USA) was instrumental in popularizing micropropagation for horticultural species. Micropropagation has now become an industrial technology, and several commercial companies round the world, including India, are using it for clonal propagation of horticultural and forest species (Chap. 17).

In 1958, Reinert (Germany) and Steward et al. (USA) demonstrated that plant regeneration in tissue cultures could also occur via embryogene-sis. They observed differentiation of somatic embryos in the cultures of root tissue of carrot. These observations fascinated many scientists because in nature embryo formation is restricted to seeds. Backs-Hüsemann and Reinert (1970) achieved embryo formation from an isolated single cell of carrot. Somatic embryogenesis has been projected as the future method of rapid cloning of plants because: (a) the embryos are bipolar with root and shoot primordia, and (b) they can be converted into synthetic seeds by encapsulation in biodegradable substances for direct field planting (Chap. 7).

GEORGES MOREL (1916-1973) FREDERICK C. STEWARD (1904-1993) HERBERT E. STREET (1913-1977) ATSUSHI KOMAMINE (1929-2011) 4 1 Historical Sketch

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By the early 1960s, methods of in vitro cul-ture were reasonably well developed, and the emphasis was shifting toward applied aspects of the technique. Cocking (1960) demonstrated that a large number of protoplasts could be isolated by enzymatic degradation of cell walls. He used culture filtrates of the fungus Myrothecium verrucaria to degrade cell walls. Takebe et al. (1968) were the first to use commercially available enzymes, cellulase, and macerozyme, to isolate protoplasts from tobacco mesophyll cells. In 1971, the totipotency of isolated plant protoplasts was demonstrated (Nagata and Takebe 1971; Takebe et al. 1970). At almost the same time, Cocking’s group in the UK achieved fusion of isolated protoplasts using NaNO3

(Power et al. 1970). Since then more efficient methods of protoplast fusion, using high pH-high Ca2+ (Keller and Melchers 1973), polyethylene glycol (Wallin et al. 1974; Kao et al. 1974), and electrofusion (Zimmermann and Vienka 1982) have been developed. These discoveries gave birth to a new field of somatic hybridization and cybridization (Chap. 14). Carlson et al. (1972) produced the first somatic hybrids between the sexually compatible parents N. glauca and N. langsdorffii. In 1978, Melchers and co-workers produced intergeneric somatic hybrids between sexually incompatible parents, potato and tomato, but the hybrids were sexually sterile. A unique application of protoplast fusion is in the production of cybrids, with novel nuclear-cytoplasmic combinations. This tech-nique has already been used to transfer male sterility inter- and intraspecifically.

In India, tissue culture started in 1957 at the Department of Botany, University of Delhi under the dynamic leadership of P. Maheshwari. The emphasis was on in vitro culture of reproductive structures (ovary, ovule, nucellus, and embryo) of flowering plants. Some pioneering contributions were made at this school. Incidentally, one of the first International Conferences on plant tissue culture was held at the Department of Botany, University of Delhi in December 1961 (see Maheshwari and Rangaswamy 1963). Prompted by her success with intra-ovarian pollination (Kanta 1960), Kanta et al. (1962) developed the technique of test tube fertilization. It involved culturing excised ovules (attached to a piece of placental tissue) and pollen grains together on the same medium; the pollen germinated and fertil-ized the ovule. Using this approach, Zenkteler and co-workers (Poland) produced interspecific and intergeneric hybrids unknown in nature (see Bhojwani and Raste 1996; Zenkteler 1999). Kranz et al. (1990) reported a major breakthrough when they electrofused isolated male and female gametes of maize and 3 years later regenerated fertile plants from the in vitro formed zygotes (Kranz and Lörz 1993). EDWARD C. COCKING (Born 1931) PANCHANAN MAHESHWARI (1904-1966) ERHARD KRANZ (Born 1947) 1 Historical Sketch 5

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In 1964, the Delhi school made another major discovery when Guha and Maheshwari demon-strated that in anther cultures of Datura innoxia the microspores (immature pollen) could be induced to form sporophytes (androgenesis). Bourgin and Nitsch (1967) confirmed the toti-potency of pollen grains, and Nitsch and Norreel (1973) succeeded in raising haploid plants from isolated microspore cultures of Datura innoxia. Production of androgenic haploids by anther or microspore culture, now reported in several crop plants, has become an important adjunct to plant breeding tools and is being widely used by plant breeders (Chap. 8). Androgenesis also provides a unique opportunity to screen gametophytic variation at the sporophytic level. For some plants, where androgenesis is difficult or not possible, haploids can be obtained by culturing unfertilized ovules or ovaries (Chap. 9). San Noeum (1976) published the first report of gynogenic haploid formation in unfertilized ovary cultures of barley.

In 1965, Johri and Bhojwani reported for the first time differentiation of triploid shoots from the cultured mature endosperm of Exocarpus cupressiformis. It provides a direct, single step approach to produce triploid plants.

Regeneration of plants from carrot cells fro-zen at the temperature of liquid nitrogen (-196°C) was first reported by Nag and Street in 1973. Seibert (1976) demonstrated that even shoot tips of carnation survived exposure to the super-low temperature of liquid nitrogen. This and subsequent successes with freeze preserva-tion of cells, shoot tips and embryos gave birth to a new applied area of plant tissue culture, called in vitro conservation of germplasm. Cul-tured shoots could also be stored at 4°C for 1–3 years. These methods are being used at several laboratories to establish in vitro reposi-tory of valuable germplasm.

The Pfizer Company made the first attempt for in vitro production of secondary metabolites on industrial scale during 1950–1960 for which Routin and Nickell (1956) obtained the first patent. Tulecke and Nickell (1956) first reported large-scale culture of plant cells in a 134 L bioreactor. Shikonin from cell cultures of Lithospermum erythrorhizon was the first in vitro produced phytochemical to be commer-cialized in 1983 by Mitsui Petrochemical Co., Japan (Curtin 1983). The other industrial com-pounds under commercial production through tissue culture are taxol and ginseng.

For long the variations observed in ploidy, morphology, pigmentation, and growth rates of cultured cells were ignored as mere abnormali-ties. Heinz and Mee (1971) published the first report of morphological variation in sugarcane hybrids regenerated from cell cultures. The agronomic importance of such variability was immediately recognized and the regenerants were screened for useful variations. During the next few years, Saccharum clones with resis-tance to various fungal and viral diseases as well as variation in yield, growth habit and sugar content were isolated (Krishnamurthi and Tlaskal 1974; Heinz et al. 1977). Larkin and Scowcroft (1981) reviewed the literature on

SIPRA GUHA-MUKHERJEE (1938-2007)

SATISH C. MAHESHWARI (Born 1933)

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spontaneous in vitro occurring variation suitable for crop improvement, and termed the variation in the regenerants from somatic tissue cultures as somaclonal variation. Evans et al. (1984) introduced the term gametoclones for the plants regenerated from gametic cells. Several soma-clones (Chap. 12) and gametoclones (Chap. 8) have already been released as new improved cultivars.

Based on his extensive studies on crown gall tissue culture, Braun (1947) suggested that prob-ably during infection the bacterium introduces a tumor-inducing principle into the plant genome. Subsequently, Chilton et al. (1977) demonstrated that the crown galls were actually produced as a result of transfer and integration of genes from the bacteria Agrobacterium tumefaciens into the plant genome, which led to the use of this bacterium as a gene transfer system in plants.

The first transgenic tobacco plants expressing engineered foreign genes were produced by Horsch et al. (1984) with the aid of A. tumefaciens.

Since 1988, biolistic gun, also called particle gun, has become a popular means to deliver purified genes into plant cells (see McCabe and Christou 1993). In 1986, Abel et al. produced the first genetically engineered plants for a useful agro-nomic trait. The list of genetically engineered varieties with useful traits has considerably enlarged, and since 1993 several transgenic vari-eties of crop plants, such as canola, cotton, maize, rice, tomato, and soybean, have been released. In 1996, nearly 5 million acres of biotech crops were sown, mainly in the USA and by 2007 these fig-ures rose to 282 million acres in 23 countries (Vasil 2008). Efforts are now being made to genetically modify plants in such a way so as to utilize them as factories for producing desired biomolecules in large quantities (Chap. 15).

These, in brief, are some of the milestones in the history of plant tissue culture. Like any other area of science, plant tissue culture started as an academic exercise to answer some basic ques-tions related to plant growth and development. However, over the years it has emerged as a tool of immense practical value. Plant tissue culture is being extensively used for clonal plant prop-agation, germplasm storage, production, and maintenance of disease-free plants and as a valuable adjunct to the conventional methods of plant improvement. Plant tissue culture tech-niques are also being extensively used in basic studies related to plant growth and development, cytodifferentiation, physiology, biochemistry, genetics, and pathology.

Plant tissue culture in India was started way back in 1957 at the Department of Botany, University of Delhi, India. Soon active centers of plant tissue culture were established at the Bose Institute, Kolkata, M.S. University, Vadodra, National Botanical Research Institute, Lucknow, and National Chemical Laboratory, Pune. The creation of the Department of Bio-technology (DBT) by the Government of India in 1986 gave a substantial boost to plant tissue culture research in this country. Many new tissue culture laboratories appeared in several tradi-tional and agricultural universities and institutes across the country. DBT supported the estab-lishment of plant tissue culture pilot plants at

ARMIN C. BRAUN (1911-1986)

MARY-DELL CHILTON (Born 1939)

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Tata Energy Research Institute, New Delhi and National Chemical Laboratory, Pune in 1989, National Research Centre for Plant Biotechnol-ogy at IARI, New Delhi, in 1985, National Facility for Plant Tissue Culture Repository at National Bureau of plant Genetic Resources (NBPGR), New Delhi in 1986 and National Gene Banks of Medicinal and Aromatic Plants at NBPGR, New Delhi, Central Institute of Medicinal and Aromatic Plants, Lucknow, Tropical Botanic Garden and Research Institute, Thiruvananthapuram, and Regional Research Laboratories, Jammu in 1993.

In 1970, International Association of Plant Tissue Culture (IAPTC) was established to promote research and development in this area, and in 1971 it started publishing ‘‘IAPTC Newsletter’’ with one or two feature articles on a current topic, forthcoming events related to PTC, list of recent publications and highlights of major developments in the area. The association organizes international conferences once in 4 years in different parts of the globe. The association was renamed in 1998 as ‘‘Interna-tional Association of Plant Tissue Culture and Biotechnology’’ and again in 2006 as ‘‘Interna-tional Association of Plant Biotechnology’’. Similarly, the Newsletter of IAPTC was renamed in 1995 as ‘‘Journal of Plant Tissue Culture & Biotechnology’’. Now it is published as a part of the journal ‘‘In Vitro Cellular and Developmental Biology – Plant’’. For more detailed history of plant tissue culture see White (1943), Krikorian and Berquam (1969), Gau-theret (1985), Bhojwani and Razdan (1996), Thorpe (2007) and Vasil (2008).

1.1 Landmarks/Milestones

1. 1902—Haberlandt presented the classic paper describing his pioneering attempt to culture isolated plant cells in a simple nutrient solution at a meeting of the Vienna Academy of Sciences in Germany.

2. 1904—Hannig initiated the work on excised embryo culture of several Crucifers.

3. 1922—Knudson demonstrated asymbiotic in vitro germination of orchid seeds. 4. 1925, 1929—Laibach demonstrated the

practical application of embryo culture to produce interspecific hybrids between sex-ually incompatible parents (Linum perenne x L. austriacum).

5. 1934—White established continuously growing cultures of tomato root tips. 6. 1937—White formulated the first synthetic

plant tissue culture medium (WM).

7. 1939—Gautheret, Nobécourt and White, independently, established continuously growing tissue cultures.

8. 1941—Van Overbeek introduced coconut water as a medium constituent by demon-strating its beneficial effect on in vitro development of immature embryos and callus formation in Datura.

9. 1946—Ball succeeded in raising whole plants from excised shoot tips of Lupinus and Tropaeolum.

10. 1947—Braun proposed the concept of tumor inducing principal (TiP) of Agro-bacterium tumefaciens responsible for autonomous growth of crown gall tissue. 11. 1950—Braun demonstrated that Ti principal

in Agrobacterium tumefaciens is transferred to plant genome naturally.

12. 1952—Morel & Martin developed the tech-nique of meristem culture of Dahlia to raise virus-free plants from infected individuals. 13. 1954—Muir et al. succeeded in inducing

divisions in mechanically isolated single cells cultured in the presence of a nurse tissue. 14. 1955—Miller et al. discovered the first cytokinin (kinetin) from autoclaved herring sperm DNA.

15. 1957—Skoog and Miller put forth the con-cept of chemical control of organogenesis (root and shoot differentiation) by manipu-lating the relative concentrations of auxin and kinetin.

16. 1958—Steward (USA) and Reinert (Germany), independently, reported the formation of embryos by the somatic cells of carrot (somatic embryogenesis).

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17. 1960—Jones et al. successfully cultured isolated single cells using conditioned medium in microchamber.

18. 1960—Bergmann developed the cell plating technique for the culture of isolated single cells.

19. 1960—Morel described a method for rapid in vitro clonal propagation of orchids (micropropagation).

20. 1960—Cocking isolated plant protoplasts enzymatically.

21. 1962—Kanta et al. developed the technique of in vitro pollination; viable seed formation by in vitro pollination of naked ovules. 22. 1962—Murashige & Skoog formulated the

most widely used plant tissue culture med-ium (MS).

23. 1964—Guha and Maheshwari produced the first androgenic haploid plants of Datura by anther culture.

24. 1965—Johri and Bhojwani demonstrated the totipotency of triploid endosperm cells. 25. 1965—Vasil and Hildebrand achieved regeneration of full plants starting from isolated single cells of tobacco.

26. 1966—Kohlenbach succeeded in inducing divisions in isolated mature mesophyll cells of Macleaya cordata which later differenti-ated somatic embryos.

27. 1970—Power et al. published the first report of chemical fusion of plant protoplast. 28. 1970—Establishment of International

Asso-ciation of Plant Tissue Culture (IAPTC). 29. 1971—Heinz and Mee reported somaclonal

variation in the regenerants from callus cultures of sugarcane.

30. 1971—Takebe et al. achieved plant regen-eration from isolated protoplasts of tobacco. 31. 1971—Newsletter of IAPTC launched. 32. 1972—Carlson et al. produced the first

somatic hybrids by the fusion of isolated protoplasts of Nicotiana glauca and N. langsdorffii.

33. 1973—Nitsch and Norreel succeeded in producing haploid plants from isolated microspore cultures of tobacco.

34. 1973—Nag and Street succeeded in regen-eration of plants from carrot cells frozen in liquid nitrogen (-196°C).

35. 1974—Zaenen et al. identified Ti plasmid as the causative factor of Agrobacterium tum-efaciens for crown gall formation.

36. 1974—Kao et al. and Walin et al. intro-duced PEG as a versatile chemical for the fusion of plant protoplasts.

37. 1974—Reinhard reported biotransformation by plant tissue cultures.

38. 1976—Seibert reported regeneration of shoots from cryopreserved shoot.

39. 1976—San Noeum reported the develop-ment of gynogenic haploids from the cul-tured unfertilized ovaries of barley. 40. 1977—Chilton et al. demonstrated that only

a part of the Ti plasmid of A. tumefaciens is responsible for crown gall formation. 41. 1984—Horsch et al. produced the first

transgenic plants of tobacco by co-culture of leaf discs with Agrobacterium tumefaciens. 42. 1986—Abel et al. produced the first trans-genic plants with useful agronomic traits. 43. 1987—Sanford et al. invented the biolistic

method of direct gene transfer into plant cells. 44. 1987—Fujita and Tabata developed com-mercial process for the production of shikonin by cell cultures of Lithospermum erythrorhizon.

45. 1993—Kranz et al. reported regeneration of full plants from in vitro fertilized eggs of maize (In Vitro Fertilization).

46. 1994—Holm et al. succeeded in raising full plants from excised in situ fertilized eggs (zygotes) of barley.

47. 1995-To date; the existing in vitro techniques were refined to enhance their efficiency and were applied to increasing number of plant species with different objectives.

48. 1995—IAPTC Newsletter developed into Journal of Plant Tissue Culture and Biotechnology.

49. 1998—IAPTC renamed as International Association of Plant Tissue Culture and Biotechnology (IAPTC & B).

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50. 2006—IAPTC & B renamed as International Association of Plant Biotechnology (IAPB).

2 General Requirements and

Techniques

2.1 Introduction

A plant tissue culture laboratory, whether for research or for commercial purpose, should pro-vide certain basic facilities, such as (i) washing and storage of glassware, plasticware and other labwares, (ii) preparation, sterilization and stor-age of nutrient media, (iii) aseptic manipulation of plant material, (iv) maintenance of cultures under controlled conditions of temperature, light and humidity, (v) observation of cultures and (vi) hardening of in vitro developed plants. The extent of sophistication in terms of equipment and facilities depends on the need and the funds available. Therefore, establishment of a new tis-sue culture facility requiring ingenuity and careful planning.

2.2 Requirements

2.2.1 Structure and Utilities

The construction of a laboratory from scratch is a costly affair but there is considerable scope for maneuverability with the design at the concep-tional stage and in the selection of construction material. To begin with, a commercial laboratory is best housed in a pre-existing building with suitable modifications. After carefully examining the economic feasibility of the venture an inde-pendent facility may be erected. More often than not, for research work the tissue culture laboratory

is carved out of the existing infrastructure, and several facilities/equipments are shared with other laboratories. A research facility should have at least four rooms: (i) Washing Room, for glass-ware washing, storage and autoclaving (ii) Media Room, for media preparation (iii) Sterile Area, for aseptic manipulation and (iv) Growth/Culture Room, to maintain cultures under suitable envi-ronmental conditions. The culture room should also have a working table, a stereoscopic micro-scope and a good light source, preferably cool light (fiber optics), for observing cultures. The sterile transfer cabinets could be placed in the culture room or in a specially designed transfer room. In many research laboratories it is kept in an undisturbed area of a general lab.

In case the facility needs to be constructed, especially for a commercial setup, it would be desirable to locate the unit away from the city to avoid heavy pollution and vehicular vibrations. However, this may require transportation of the personnel. The location of the laboratory should not be near fields to avoid spurts of infection by the combines and threshers during the harvest seasons. The facility needs to be adequately protected from rains and winds as these carry spores, mites and thrips. Thermal insulation of the facility to conserve energy is another aspect requiring proper thought. One way is to have the transfer area and the growth rooms below ground level. In that case care must be taken to protect the lab from seepage and provide ade-quate ventilation. Alternately, these two rooms could have a double wall or built of hollow

S. S. Bhojwani and P. K. Dantu, Plant Tissue Culture: An Introductory Text, DOI: 10.1007/978-81-322-1026-9_2,Ó Springer India 2013

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bricks with air trapped in between, which could be vented during summers.

A tissue culture facility requires large quan-tities of good quality water. At the designing stage itself adequate attention should be paid to the source of water and waste-water disposal, especially where sewer facilities are not avail-able, keeping in view the local municipal rules for health and environment.

A tissue culture unit must have power backup to save cultures from getting contaminated in the event of power failure or load-shedding from the mains during aseptic manipulations. Valuable cultures may be lost because of temperature shocks in the growth room during electricity breakdowns/shutdown. The generator may be fitted with a self-starting switch.

It is of paramount importance that a tissue culture laboratory is clean and movement of materials from one area to another occurs with minimal backtracking. These aspects should be the guiding principle while designing the layout plan of various rooms, pass-through windows, doors and hallways. It is necessary and desirable to isolate the ‘clean area’, comprising of transfer room and growth room from rest of the ‘unclean area’ and it should be treated as ‘restricted area’, out of bounds for visitors and outsiders. In the passage between these two areas, especially in a commercial set-up, one is required to wash hands and feet and wear sterilized overcoats and headgear before entering the ‘restricted area’. Generally, high standards of sanitation need to be maintained and these have to be more strin-gent where dust, pollen and small insects abound in the environment. It is a good idea to have paved pathways and shrubs around. High levels of cleanliness and freedom from extraneous materials could be achieved by having positive air pressure, at least in the ‘clean area’. Depending on the necessity, a Class 1,000 or Class 10,000 standard should be maintained for the clean room. For the movement of material in (sterilized medium, instruments, water, etc.) and out of (glassware, old and infected cultures, tissue culture produced plants for hardening, etc.) the ‘clean area’ a window with double door

hatch should be provided to maintain high asepsis in the ‘clean area’.

As far as possible indigenously available construction material, equipment, apparatus, and instruments should be used for cost effectiveness and ease of maintenance. Innovativeness and indigenous fabrication will go a long way in reducing the costs.

2.2.2 Washing Room

Depending on the availability of funds and space the washing and sterilization areas may be in separate rooms or in a common room. In either case, the washing area should have adequate supply of good quality hot and cold running water and an acid and alkali resistant big sink. Adequate steel or plastic buckets and tubs are required for soaking culture vials and other labwares used in medium preparation. Brushes of various sizes and shapes are essential for cleaning glassware, while it is optional to have a dish washing machine. For a commercial set-up an industrial dish washer is desirable. The media room should have a hot air cabinet to dry the washed labware, an oven for dry sterilization, and a dust proof cupboard for storage of plastic and glass-ware. When washing is done in media room a temporary partition can be erected between the two areas to prevent splashing of soap solution and any other interference in the two activities. Alternately, timings of the two activities could be staggered. Where the auto-clave is to be housed within the media room an isolated area with provision for ventilation through an exhaust should be chosen.

Even if good quality water is available it cannot be used for final washing of labwares or for medium preparation as it contains impurities such as inorganic and organic compounds, dis-solved gases, particulate debris and microor-ganisms. Water could be purified through distillation, deionization or reverse osmosis. Sometimes a combination of two or more is required. Water purity is measured in terms of resistivity (ohms cm-1) or its reciprocal, i.e.,

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conductivity (mhos cm-1). Water for tissue culture should ideally have a conductivity of 5.0 lmhos cm-1although a conductivity level up to 15 lmhos cm-1 is acceptable. Deionized water may be used for teaching laboratories or for rinsing labware but for research and commercial purposes, water distillation apparatus, a reverse osmosis unit or a Mili-Q purification system needs to be installed. The choice between the three is one of quality of final water, speed of production and cost. For a research laboratory a glass distillation unit with a handling capacity of 1.5 to 2 L h-1of water should be sufficient. For a commercial set-up or where high purity water is required a Mili-Q purification system that can provide 90 L h-1 may be used. Proper storage tanks should be provided for the purified water.

2.2.3 Media Room

The media room is the kitchen of the tissue culture facility. The media room is provided with a working table in the centre and benches along the wall, the tops of which are either covered with granite or laminated board (Fig.2.1). The tables and benches should be at a

height suitable for working while standing and the space below them could be fitted with drawers and cupboards for storage purposes. The benches are required for keeping balances, pH meter, magnetic stirrers, hot plates etc. A top loading electronic balance with tare for weigh-ing large quantities and an analytical balance for small quantities of chemicals must be provided. The balances should be isolated in a small chamber if the media room also houses the autoclave. In a large commercial laboratory it will be of help to have an automatic media dispenser.

For short term storage of certain chemicals, plant materials, and stock solutions a refrigerator and a deep freeze are required. These could be kept in the corridor if sufficient space in the room is not available. A single electrode pH meter that can read conductivity also should be provided. For filter sterilization of medium or solutions of thermolabile compounds an aspira-tor or vacuum pump may be required. For steam sterilization an autoclave or a domestic pressure cooker, depending on the quantity to be steril-ized, is needed.

For emergencies a fire extinguisher and a first aid kit should be kept in this room.

Fig. 2.1 Media preparation

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2.2.4 Glassware/Plasticware

In a tissue culture laboratory culture vessels (Fig.2.2) are required in bulk. Depending on the type of work, adequate supplies of these should be maintained. For standard tissue culture work rimless test tubes (25 9 150 mm) are widely used (Fig.2.2a). The culture tubes are important for culture initiation and establishment even in a commercial set-up. For further mass multipli-cation larger containers such as jam bottles or other wide mouthed bottles are required. Erlen-meyer flasks have also been used as culture vessels (Fig.2.2b). Only borosilicate or Pyrex glassware should be used.

Plastic culture vials, autoclavable and prester-ilized, have replaced glass culture vials to a large extent. A wide range of presterilized, disposable culture vials made of clear plastic, especially designed for protoplast, cell, tissue and organ culture, are available in the market under different brand names. The presterilized plastic Petri dishes (Fig.2.2c), jars (Fig.2.2d), screw cap bottles, and various cell culture plates come with their clo-sures. For culture tubes and flasks, traditionally, non-absorbent cotton plugs wrapped in a single layer of cheesecloth have been used as a closure. Autoclavable, transparent polypropylene caps with a membrane built into the top are also avail-able (KimKaps, Kimble, Division of Owens, IL). Cotton plugs provide excellent aeration but the medium dehydrates very fast. On the other hand,

polypropylene caps reduce the rate of medium desiccation but increase moisture and gaseous accumulation within the container. However, it is important to ensure that the closure allows proper aseptic aeration and does not inhibit the growth of culture materials. In this regard it may be men-tioned that Parafilm/Nescofilm, commonly used to seal Petri dishes, releases butylated hydroxytolu-ene, which is toxic to the cultured plant material (Selby et al. 1996). Alternately, one can use cling film for sealing, as 2-ethyl-1-hexanol released by it is not inhibitory to culture material.

Now it is possible to buy culture vessels made of different synthetic materials. Culture vessels made of polypropylene transmit 65 % light and those made of polycarbonate transmit almost 100 % light. Gas permeable fluorocarbonate vessels are available for use with plant materials that are sensitive to gas build up within the culture vials (Kozai 1991).

Besides culture vials, various other glass-/ plasticware such as beakers, measuring cylin-ders, pipettes, etc. of various sizes are required for media preparation.

2.2.5 Transfer Room

In research laboratories the transfer hoods are placed in the growth/culture room or even in a quite corner of a general laboratory. However, in a commercial facility it is necessary to have separate transfer and growth room(s). There are

Fig. 2.2 Culture vials. a Culture tube with polypropylene cap. b Flask. c Petri plate. d Plastic jar (Magenta box)

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no special requirements for a transfer room except that a high degree of cleanliness and a worker friendly environment is to be main-tained. Transfer hoods are placed in this room to carry out aseptic manipulations. Often the cul-ture medium to be used is also stored here although, where possible, a separate store room within the ‘clean area’ is demarcated for this purpose. To transport culture medium or cultures in and out of the transfer room, trolleys with one or more shelves are helpful. These trolleys may also serve as side benches for the operator to hold extra culture medium, stock cultures, and/ or freshly raised cultures until transferred to the growth room. Since fire/heat is constantly used in the transfer hoods it is advisable to keep a fire extinguisher in this area.

2.2.6 Growth Room

The inoculated culture vials are transferred for incubation to a growth room with controlled temperature and light conditions. It is of para-mount importance to maintain cleanliness in this area. It can be achieved by having positive air pressure in the ‘clean area’ or an overhead air-curtain at the entry to remove surface dust. This room should have one door, and the windows should be avoided to prevent external light from interfering with the internal light cycle. Desir-ably, the junction of walls should be rounded rather than angular to prevent cobwebs. The wall paints should be of semi or high-gloss with a linoleum floor to withstand repeated cleaning. Tiller et al. (2002) reported that the polymer Hexyl-PVP when coated on a glass surface kil-led 99 % of harmful bacteria.

Growth rooms being closed units require devices to control temperature and light. Tem-perature is controlled by air conditioning. Tower air conditioning is an expensive proposition, but if that is the chosen option then adequate pre-cautions should be taken to filter the cooled air to prevent dust, spores etc. from reaching the ‘clean area’. With this type of air conditioning it is easy to maintain positive air pressure. In

research laboratories generally window mounted or split air conditioners are used for cooling during summer and hot air blower for heating during winter. The air conditioners and heaters are hooked to temperature controllers to main-tain the temperature at 25 ± 2°C. For higher or lower temperature treatments, special incubators with built in fluorescent lights can be used. These incubators may be kept even outside the growth room with suitable measures to prevent anyone from tampering with the settings.

Generally, cool white fluorescent lamps with electronic ballasts are used for growth rooms because of the uniform light intensity they emit. Cultures are normally maintained in diffuse light (3000–5000 lux). The low light intensity can be achieved in 3.5 feet wide shelves by installing three tubes one foot above the surface of the shelf. A sheet of aluminium foil or coat of aluminium paint provided above the tubes maximizes light intensity below. Some provision should also be made for growing cultures in total darkness or under high light intensities. Automatic timers are used to regulate the photoperiod.

If the relative humidity in the growth room falls below 50 %, humidifiers are required to be used to prevent medium from drying rapidly. Dehumidi-fiers may be required when the humidity is very high, particularly during the rainy season, to pre-vent cotton plugs from becoming damp or con-densing of water droplets, as both may increase chances of contamination of cultures.

Culture vials in the growth room are main-tained on specially designed shelving unit that are either stationary or moveable. Stationary shelves may be fixed on walls of the room, or could be fitted into angular iron frames to form culture racks (Fig.2.3) that are placed conveniently in the room. Alternately, the racks could be on roller coaster wheels that allows efficient use of space. Open shelves are generally preferred because of better air circulation. The shelves can be made of plywood board or rigid wire mesh. Each shelf is provided with a separate set of fluorescent tubes. In case the cultures are to be maintained under different photoperiod and temperature regimes it is advisable to have more than one growth room.

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Culture flasks, jars and Petri dishes can be placed directly on the shelves or in trays of suitable size, while culture tubes require some support such as a metallic wire rack (Fig.2.3), which can hold 20–24 tubes. In commercial companies, where large quantities of culture vials have to be moved, it is not only convenient but also time saving to use autoclavable plastic or epoxy coated metallic trays for holding the culture vials. The culture vials and the trays holding them should be appropriately labeled to avoid mixing up. For transportation of culture vessels cart trolleys may be used.

The culture room should also have a shaking machine, either of the horizontal type or the rotary type, if cell suspension cultures are to be grown. Shakers with speed, temperature and light controls are also available.

2.2.7 Cold Storage

In a commercial setup it is necessary to have a cold storage maintained at 2–4°C for temperate plants and 15°C for tropical plants. These rooms are used to give treatment for breaking dormancy of some plant materials, storing of cultures to

schedule workload, maintain ‘mother’ cultures and to hold harvested plants (Mageau1991).

2.2.8 Greenhouse

In order to grow the mother plants and to accli-matize in vitro produced plants, a tissue culture laboratory should have a greenhouse made of glass, polythene or polycarbonate depending on the budgetary provisions. This facility should have a provision to maintain high humidity such as fan and pad system. It would be desirable to have a potting room adjacent to this facility. A separate autoclave might be required in this area if one wants to sterilize the potting mixture. In a commercial laboratory provision for cer-tain other rooms such as, a general storage, and employee’s tea room, an administrative office and shipping and receiving centre should be made.

2.3 Techniques

This section deals with the basic techniques of maintaining cleanliness and asepsis in the labo-ratory and in the cultures.

Fig. 2.3 a An illuminated culture trolley with six shelves holding culture tubes arranged in metallic racks. b An enlarged portion of a trolley as in (a), with cultures in screw cap bottles

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2.3.1 Glassware and Plasticware

Washing

All glassware and plasticware, except pre-sterilized ones, should be thoroughly washed when using for the first time. As a normal practice, the apparatus is soaked overnight in a standard laboratory detergent and scrubbed with a bottle brush manually or by a machine. These are then rinsed under tap water followed by a rinse in distilled water. Dried agar can be removed by heating. The contaminated glass and plastic culture vials should be autoclaved before opening for washing or discarding, respectively, in order to minimize the spreading of bacterial and fungal contaminants in the laboratory. The washed apparatus are placed in wire baskets or trays to allow maximum drainage and dried in a hot air cabinet at about 75°C and stored in a dust proof cupboard. For transportation of washed labware from washing area suitable trays and mobile carts can be used.

2.3.2 Sterilization

Whether it is labware or culture medium, plant material or environment in the laboratory, instruments used for culture or the operator himself, all are sources of infection. The tissue culture medium being rich in sugar and other organic and inorganic nutrients supports good growth of microorganisms, such as fungi and bacteria. On reaching the medium the microor-ganisms may grow faster than the plant tissues, finally killing them. The microbes may also secrete toxic wastes into the medium inhibiting growth of cultured tissues. It is, therefore, absolutely essential to maintain a completely aseptic environment inside culture vessels. As a rule, plant tissue culture laboratory facilities should not be shared with microbiologists and pathologists, and contaminated vessels should be removed as soon as detected.

The various sources of contamination and measures to guard cultures against them are discussed in the following pages.

(i) Glassware and Plasticware. Culture vials are a major source of contamination, more so, if these have been in long use. The glass culture vials may be dry sterilized before pouring the medium to kill such bacteria, which might withstand autoclav-ing. Culture vials are generally sterilized together with the culture medium. For pre-sterilized medium the culture vials with proper closure may be sterilized by auto-claving or dry heating in an oven at 160–180°C for 3 h. For dry heating the oven should have a fan mounted inside for better circulation of hot air. It is important that the oven is not over loaded. The glassware should be allowed to cool down before removing it from the oven. Other-wise, cool air sucked from outside may expose the load to bacterial contamination and also increase the risk of cracking. Not all types of plastic labware can be heat sterilized. Only polypropylene, polymeth-ylpentene, polyallomer, Tefzel ETFE and Teflon FEP may be repeatedly autoclaved at 121°C. Sterilization cycle for polycar-bonate vials should be limited to only 20 min as it shows loss of mechanical strength on repeated autoclaving. Polysty-rene, polyvinyl chlorides, styrene acrylo-nitrile, are not autoclavable at all.

(ii) Culture Medium. The tissue culture medium, as stated earlier, is not only a source of contaminants but also supports their luxuri-ant growth. Therefore, it must be sterilized properly. Mostly, the culture medium is sterilized by autoclaving. Autoclave (Figs.2.4, 2.5) is an apparatus in which sterilization is done by steam heating under pressure. The culture vials containing med-ium and closed with a suitable bacteria-proof closure are autoclaved at 15 psi and 121°C for 15–40 min from the time the medium reaches the required temperature and pres-sure. Care should be taken to cover cotton plugs and other things that might get wet with aluminium foil before autoclaving. For sterilization of small quantities of medium a

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Fig. 2.4 A horizontal autoclave (a) and a small vertical autoclave (b)

Fig. 2.5 Working diagramme of a horizontal autoclave

Plant Tissue Culture.pdf

Plant Tissue

Culture:

An Introductory

Text

Sant Saran Bhojwani

Prem Kumar Dantu

Plant Tissue Culture:

An Introductory Text

Sant Saran Bhojwani

Prem Kumar Dantu

Plant Tissue Culture:

An Introductory Text

Dedicated to the most Revered

Dr. M. B. Lal Sahab (1907–2002)

D.Sc. (Lucknow), D.Sc. (Edinburgh),

the visionary Founder Director of the

Dayalbagh Educational Institute, for

the inspiration and strength to

undertake and complete the task of

writing this book

Preface

Contents

About the Book

1

Historical Sketch

1.1

Landmarks/Milestones

Suggested Further Reading

2

General Requirements and

Techniques

2.1

Introduction

2.2

Requirements

2.2.1

Structure and Utilities

2.2.2

Washing Room

2.2.3

Media Room

2.2.4

Glassware/Plasticware

2.2.5

Transfer Room

2.2.6

Growth Room

2.2.7

Cold Storage

2.2.8

Greenhouse

2.3

Techniques

2.3.1

Glassware and Plasticware

Washing

2.3.2

Sterilization